During the last years, small size metal particles, in particular colloidal gold particles, have increasingly been used for the detection and/or quantitative determination of specific binding agents and/or their corresponding bindable substances. Usually, such bindable substances are of biological origin and their determination by means of metal labelling has proven useful in various areas of biochemistry, pharmacology, cytology and histology. The concerned techniques have found widespread use and are particularly attractive for diagnostic purposes.
In principle, the use of colloidal metal particles as markers is based on the fact that the specific binding agent or any substance bindable thereby, when brought into contact with the colloidal metal particles under appropriate conditions, do strongly absorb thereon without loosing their affinity for their binding or bindable counterpart. Mostly, the relationship between a specific binding agent and its corresponding bindable substance(s) will be of the type, antigen-antibody, antibody-antigen, protein-protein, protein-ligand, receptor-ligand or nucleic acid-complementary nucleic acid. Hence, the thus labelled specific binding agents or bindable substance, when allowed to interact with their counterparts will attach their label to the complex formed during the interaction and consequently the detection thereof can easily be performed making use of the properties of the colloidal metal particles.
Depending on the circumstances, colloidal metal particles can be detected, e.g., by direct visual examination, by microscopic or spectrofotometric techniques. A description of "the immunogold staining (IGS) technique", "the sol particle immuno assay (SPIA) technique" of specific applications and improvements thereof can be found in, e.g., U.S. Pat. Nos. 4,313,734, 4,446,238, 4,420,558, 4,775,636, and 4,752,567, and in IBRO handbook series, Wiley, N.Y., 1983, pages 347 to 372.
So far colloidal metal markers with a wide variation in particle sizes have been prepared. Typical examples are described in Techniques in Immunochemistry Vol. 2 (1983) and in the Journal of Histochemistry and Cytochemistry 35, 1191-1198 (1987) and the references described therein. Usually they are applied in a size range between 5 and 20 nm, however some small size colloidal metal markers of about 2.6 nm have been described in Histochemistry (1985) 83: 409-411. Detection systems based on small colloidal metal markers have a number of advantages over specific binding agents marked with larger metal particles. More particularly they give a higher labelling density and offer an increase in resolution for Electron Microscopy. However, detection systems based on these small colloidal metal particles have hardly been studied due to their instability and lack of reproducibility. It is therefore an object of the invention to provide reproducible detection systems based on stable and bio-compatible ultra small metal particles.